Bile Duct Ligation Induces ATZ Globule Clearance in a Mouse Model of α-1 Antitrypsin Deficiency

Abstract

α-1 Antitrypsin deficiency (A1ATD) can progress to cirrhosis and hepatocellular carcinoma; however, not all patients are susceptible to severe liver disease. In A1ATD, a toxic gain-of-function mutation generates insoluble ATZ "globules" in hepatocytes, overwhelming protein clearance mechanisms. The relationship between bile acids and hepatocytic autophagy is less clear but may involve altered gene expression pathways. Based on previous findings that bile duct ligation (BDL) induces autophagy, we hypothesized that retained bile acids may have hepatoprotective effects in PiZZ transgenic mice, which model A1ATD. We performed BDL and partial BDL (pBDL) in PiZZ mice, followed by analysis of liver tissues. PiZZ liver subjected to BDL showed up to 50% clearance of ATZ globules, with increased expression of autophagy proteins. Analysis of transcription factors revealed significant changes. Surprisingly nuclear TFEB, a master regulator of autophagy, remained unchanged. pBDL confirmed that ATZ globule clearance was induced by localized stimuli rather than diet or systemic effects. Several genes involved in bile metabolism were overexpressed in globule-devoid hepatocytes, compared to globule-containing cells. Retained bile acids led to a dramatic reduction of ATZ globules, with enhanced hepatocyte regeneration and autophagy. These findings support investigation of synthetic bile acids as potential autophagy-enhancing agents.

Figure 1

Bile duct ligation induces clearance of ATZ globules in PiZZ mouse liver. (A) Serum ALT, alkaline phosphatase, and total bilirubin levels at days 3, 7, and 14 after BDL or sham operations. (B) Low-power views of PASD stains show dramatic clearance of ATZ globules from the liver lobe (magnification: 40×). (C) Quantitative results of ATZ globule morphometry showing percent of PASD globules present in the tissue section. (D) PASD stain showing “cleared” region after 14d BDL (magnification: 100×). Arrow points to residual GC hepatocytes intermixed within areas of ductal proliferation.

Figure 2

Bile duct ligation induces an early regenerative response in GD hepatocytes 3 days after surgery. (A) Ki-67 immunohistochemistry costained with PASD shows regenerating Ki-67⁺ GD hepatocytes (arrows). Inset shows a rare mitosis within a repopulating cluster of GD cells in sham control. (B) Activated caspase 3 immunohistochemistry costained with PASD does not show significant apoptosis in GC hepatocytes after BDL (magnification: 200×). (C) Quantitative immunofluorescence data for the cell proliferation marker Ki-67 and anti-human A1AT. Graph depicts a small subset of percentage of Ki-67⁺ cells colocalizing with ATZ GC cells (normalized to total number of cells).

Figure 3

Bile duct ligation induces increased expression of autophagic proteins in PiZZ mouse liver. (A, B) Western blots of total liver tissue lysates showing increased Atg5, Atg12, and LC3A/B after BDL compared to sham livers. Ponceau is shown to confirm equal loading, since cytosketetal changes can affect both actin and tubulin in PiZZ mouse liver. (C) Confocal immunofluorescence microscopy showing the majority of LC3B (green) does not colocalize with GC cells labeled with anti-human A1AT (red) antibody (magnification: 100×). (D) Quantitative immunofluorescence data for LC3 and anti-human A1AT. Graph depicts a small subset of percentage of LC3⁺ cells colocalizing with ATZ GC cells (normalized to total number of cells).

Figure 4

Bile duct ligation induces transcriptional reprogramming in PiZZ mouse liver. (A, B). Western blot of nuclear proteins showing decreased nuclear C/EBP-α and C/EBP-β isoforms in BDL livers compared to sham-operated and wild-type (WT) control liver. Nuclear FXR is decreased after BDL at later time points. Nuclear TFEB is not significantly changed in BDL and sham livers. TATA-binding protein (TBP) serves as the nuclear protein-loading control.

Figure 5

Partial bile duct ligation induces localized ATZ globule clearance in PiZZ mouse liver. (A) Serum ALT, alkaline phosphatase, and total bilirubin levels in mice 14 days after pBDL are comparable to age- and sex-matched unoperated PiZZ (control) mice. As expected, biochemical parameters in BDL mice (shown in Fig. 1A) are significantly worse compared to pBDL values. (B) Quantitative results of ATZ globule morphometry showing decreased percentage of PASD globules present in the ligated left lobe (pBDL) compared to the unligated right control lobe. (C) PASD staining showing the cleared areas of ATZ globules in pBDL (ligated left lobe, right) compared to control (unligated right lobe, left). Arrows point to residual GC hepatocytes intermixed within areas of ductal proliferation, similar to total BDL (magnification: 200×).

Figure 6

Relationship of ATZ globule clearance to regeneration in partial bile duct ligation. (A) Ki-67 immunohistochemistry costained with PASD shows persistence of regenerating Ki-67⁺ GD hepatocytes 14 days after pBDL (right, arrows). (B) Activated caspase 3 immunohistochemistry costained with PASD does not show significant apoptosis in GC hepatocytes 14 days after pBDL (right) compared to unligated right control lobe (left, arrows; magnification: 200×).

Figure 7

Relationship of ATZ globule clearance to autophagy in partial bile duct ligation. (A) Confocal immunofluorescence microscopy showing the cleared areas of red ATZ globules in pBDL lobe (right, arrow) compared to unligated control lobe (left). *Residual GC hepatocytes intermixed within areas of ductal proliferation (magnification: 100×). (B) Increased LC3B staining (green, right, arrow) in GD hepatocytes in the ligated pBDL lobe (magnification: 200×).